JP7037198B2 - Prophylactic and / or therapeutic agents for autoimmune diseases, as well as vaccines - Google Patents
Prophylactic and / or therapeutic agents for autoimmune diseases, as well as vaccines Download PDFInfo
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- JP7037198B2 JP7037198B2 JP2019502986A JP2019502986A JP7037198B2 JP 7037198 B2 JP7037198 B2 JP 7037198B2 JP 2019502986 A JP2019502986 A JP 2019502986A JP 2019502986 A JP2019502986 A JP 2019502986A JP 7037198 B2 JP7037198 B2 JP 7037198B2
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Description
本発明は、自己免疫疾患の予防および/または治療剤、並びに、自己免疫疾患の予防用および/または治療用のワクチンに関する。 The present invention relates to prophylactic and / or therapeutic agents for autoimmune diseases and vaccines for the prevention and / or treatment of autoimmune diseases.
免疫系は、本来、外界からの有害な異物の侵入に対する生体の防御機構として存在するものである。しかし、時にはこの免疫系の働きが、結果的に生体に有害であることがある。生体は、外界からの異物に対してのみならず、自己の成分に対しても免疫応答を起こすことが知られており、自己免疫現象と呼ばれている。そして、自己免疫によってある種の病態が生じた疾患は、自己免疫疾患と呼ばれている。 The immune system originally exists as a defense mechanism of a living body against the invasion of harmful foreign substances from the outside world. However, sometimes this function of the immune system is harmful to the living body as a result. It is known that a living body causes an immune response not only against foreign substances from the outside world but also against its own components, which is called an autoimmune phenomenon. A disease in which a certain condition is caused by autoimmunity is called an autoimmune disease.
自己免疫疾患は、全身性の疾患であるが、臓器特異性のある疾患(臓器特異的自己免疫疾患)と、臓器特異性のない疾患(臓器非特異的自己免疫疾患)との2つに大別される。これら自己免疫疾患の多くは、組織に病変が認められるとともに、組織傷害を伴うものである。 Autoimmune diseases are systemic diseases, but there are two major types: organ-specific diseases (organ-specific autoimmune diseases) and non-organ-specific diseases (organ-non-specific autoimmune diseases). Be separated. Many of these autoimmune diseases have tissue lesions and are associated with tissue damage.
臓器非特異的自己免疫疾患である全身性エリテマトーデス(SLE)は、抗dsDNA抗体などの自己抗体の産生の増加に起因することが知られているが、SLEを引き起こす過程の分子生物学的な研究、多様な病態との関連の解明、さらには治療法の開発が期待されている。 Systemic lupus erythematosus (SLE), an organ-nonspecific autoimmune disease, is known to be due to increased production of autoantibodies such as anti-dsDNA antibodies, but molecular biological studies of the processes that cause SLE. , Elucidation of the relationship with various pathological conditions, and development of treatment methods are expected.
実験動物を同一の抗原で繰り返して免疫し続けると、免疫応答は極期をむかえ、やがて疲弊する。その結果として、組織傷害を伴う種々の自己免疫疾患(病態)が生じることが知られている(例えば、特許文献1参照および非特許文献1参照)。
When the experimental animal is repeatedly immunized with the same antigen, the immune response reaches the extreme stage and eventually becomes exhausted. As a result, it is known that various autoimmune diseases (pathological conditions) accompanied by tissue injury occur (see, for example,
自己免疫疾患による組織傷害に関する研究は現在進みつつあり、組織傷害に、抗原提示細胞による抗原のクロスプレゼンテーションが関わっていることが明らかにされている。具体的には、(1)自己免疫疾患を患うと、樹状細胞における抗原のクロスプレゼンテーションが増強されること、(2)樹状細胞におけるクロスプレゼンテーションが増強されるとCD8+T細胞が活性化し、当該CD8+T細胞によって細胞傷害が誘導されること、が明らかにされている(例えば、特許文献2および非特許文献1参照)。Research on tissue damage caused by autoimmune diseases is currently underway, and it has been clarified that the cross-presentation of antigens by antigen-presenting cells is involved in tissue damage. Specifically, (1) suffering from autoimmune disease enhances antigen cross-presentation in dendritic cells, and (2) enhances cross-presentation in dendritic cells activates CD8 + T cells. , It has been clarified that the CD8 + T cells induce cell damage (see, for example,
本発明者らは、マウスに対して抗原による免疫操作を繰り返すことによって、自己応答性・自己抗体誘導性CD4 T細胞(autoantibody-inducing CD4 T cells;aiCD4 T細胞)という新たなタイプのT細胞が末梢にて生成され、aiCD4 T細胞が多様な自己抗体を誘導するとともに、細胞傷害性T細胞(CTL)を成熟させて種々の臓器障害を惹起して、SLEを発症させる、ということをこれまでに報告し(非特許文献1)、さらに、aiCD4 T細胞が、PD-1+CD45RBlow122low(PD-1+CD45RBlo122lo)のCD4 T細胞亜集団に存在すること、この細胞亜集団をナイーブなマウスに養子移入すると、移入2週間後のレシピエントマウスの血清中に自己抗体が観察されることを見出している(非特許文献2参照)。By repeating immune manipulation with antigens against mice, the present inventors have created a new type of T cells called autoantibody-inducing CD4 T cells (aiCD4 T cells). So far, aiCD4 T cells, which are produced in the periphery, induce various autoantibodies and mature cytotoxic T cells (CTLs) to cause various organ damages and cause SLE. (Non-Patent Document 1), and further, that aiCD4 T cells are present in the CD4 T cell subpopulation of PD-1 + CD45RB low 122 low (PD-1 + CD45RB lo 122 lo ), this cell subpopulation. It has been found that when the cells are adopted into naive mice, autoantibodies are observed in the sera of
更に、本発明者らは、自己免疫疾患の発症の原因となる細胞をより簡便に特定するためのマーカーを見出し、これに基づいて自己免疫疾患の客観的かつ正確な診断を可能とする技術の開発に成功している(特許文献3参照)。 Furthermore, the present inventors have found a marker for more easily identifying a cell that causes the onset of an autoimmune disease, and based on this, a technique that enables objective and accurate diagnosis of an autoimmune disease. It has been successfully developed (see Patent Document 3).
しかしながら、自己免疫疾患の予防および/または治療に用いられる薬剤に関しては、いまだ十分とはいえず、新規薬剤の更なる開発が望まれていた。 However, the drugs used for the prevention and / or treatment of autoimmune diseases are not yet sufficient, and further development of new drugs has been desired.
本発明は、自己免疫疾患の予防および/または治療に用いられる新たな薬剤を提供することを目的とする。 It is an object of the present invention to provide a novel agent used for the prevention and / or treatment of autoimmune diseases.
本発明者は、鋭意検討した結果、抗DOCK8抗体を用いることによって、自己免疫疾患の症状を改善できることを見出し、本発明を完成させるに至った。本発明者が見出した知見は、DOCK8タンパク質が自己免疫疾患の発症過程に関与していることを示唆する知見であり、当該分野にとって、驚くべき知見であった。 As a result of diligent studies, the present inventor has found that the symptoms of autoimmune diseases can be improved by using an anti-DOCK8 antibody, and has completed the present invention. The findings found by the present inventor suggest that the DOCK8 protein is involved in the pathogenic process of autoimmune diseases, which is a surprising finding for the field.
上記の課題を解決するために、本発明の一態様に係る自己免疫疾患の予防および/または治療剤は、抗DOCK8抗体を有効成分として含有することを特徴としている。 In order to solve the above problems, the prophylactic and / or therapeutic agent for an autoimmune disease according to one aspect of the present invention is characterized by containing an anti-DOCK8 antibody as an active ingredient.
上記の課題を解決するために、本発明の一態様に係る自己免疫疾患の予防用および/または治療用のワクチンは、DOCK8タンパク質、DOCK8タンパク質の部分配列、または、DOCK8タンパク質若しくはDOCK8タンパク質の部分配列の発現ベクターを有効成分として含有していることを特徴としている。 In order to solve the above-mentioned problems, the vaccine for preventing and / or treating an autoimmune disease according to one aspect of the present invention is a DOCK8 protein, a partial sequence of the DOCK8 protein, or a partial sequence of the DOCK8 protein or the DOCK8 protein. It is characterized by containing the expression vector of the above as an active ingredient.
本発明の一態様によれば、自己免疫疾患の予防および/または治療を行うことができる。 According to one aspect of the present invention, autoimmune diseases can be prevented and / or treated.
本発明の一実施形態について以下に説明するが、本発明はこれに限定されるものではない。本発明は、以下に説明する各構成に限定されるものではなく、特許請求の範囲に示した範囲で種々の変更が可能であり、異なる実施形態や実施例にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態や実施例についても本発明の技術的範囲に含まれる。また、本明細書中に記載された学術文献及び特許文献の全てが、本明細書中において参考文献として援用される。また、本明細書において特記しない限り、数値範囲を表す「A~B」は、「A以上、B以下」を意図する。 An embodiment of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to the configurations described below, and various modifications can be made within the scope of the claims, and the technical means disclosed in different embodiments and examples can be used. Embodiments and examples obtained by appropriately combining them are also included in the technical scope of the present invention. In addition, all the academic and patent documents described in the present specification are incorporated as references in the present specification. Further, unless otherwise specified in the present specification, "AB" representing a numerical range is intended to be "A or more and B or less".
〔1.自己免疫疾患の予防および/または治療剤〕
上述のとおり、本発明者らは、これまで、dedicator of cytokinesis protein 8(以下、DOCK8)を発現するCD4 T細胞(DOCK8陽性CD4 T細胞(DOCK8+ CD4 T細胞ともいう。))の増加を指標として、自己免疫疾患の診断を行う技術を報告している(特許文献3)。しかしながら、このDOCK8+ CD4 T細胞がどのような機序にて自己免疫疾患の発症過程に関与しているか否かは、不明であった。そこで、本発明者らが鋭意検討した結果、驚くべきことに、DOCK8に対する抗体に、自己免疫疾患の予防/治療効果があることが分かった。なお、DOCK8+ CD4 T細胞の存在が自己免疫疾患の診断指標の一つになり得るとしても、そこから治療薬の開発には非常に大きなギャップが存在することを念のため付言しておく。[1. Preventive and / or therapeutic agents for autoimmune diseases]
As described above, the present inventors have so far indicated an increase in CD4 T cells expressing dedicator of cytokinesis protein 8 (hereinafter, DOCK8) (DOCK8-positive CD4 T cells (also referred to as DOCK8 + CD4 T cells)). (Patent Document 3) reports a technique for diagnosing autoimmune diseases. However, it was unclear by what mechanism these DOCK8 + CD4 T cells were involved in the pathogenic process of autoimmune diseases. Therefore, as a result of diligent studies by the present inventors, it was surprisingly found that the antibody against DOCK8 has a preventive / therapeutic effect on autoimmune diseases. It should be noted that even if the presence of DOCK8 + CD4 T cells can be one of the diagnostic indicators for autoimmune diseases, there is a very large gap in the development of therapeutic agents.
本実施の形態の自己免疫疾患の予防および/または治療剤は、抗DOCK8抗体を有効成分として含有するものである。 The prophylactic and / or therapeutic agent for autoimmune diseases of the present embodiment contains an anti-DOCK8 antibody as an active ingredient.
本明細書において「自己免疫疾患」とは、自己成分(例えば、免疫グロブリンなど)に対する自己抗体(例えば、抗Sm抗体、抗dsDNA抗体、リウマチ因子(RF:rheumatoid factor))が検出され、自己免疫が病態に関与している疾患を意図する。自己免疫疾患としては、臓器特異的自己免疫疾患(例えば、慢性甲状腺炎、原発性粘膜水腫、甲状腺中毒症、悪性貧血、グッドパスチャー症候群、急性進行性糸球体腎炎、重症筋無力症、尋常性天疱瘡、水疱性類天疱瘡、インスリン抵抗性糖尿病、若年性糖尿病、アジソン病、萎縮性胃炎、男性不妊症、早発性更年期、水晶体原性ぶどう膜炎、交感性脈炎、多発性硬化症、潰瘍性大腸炎、原発性胆汁性肝硬変、慢性活動性肝炎、自己免疫性溶血性貧血、発作性血色素尿症、突発性血小板減少性紫斑病、およびシェーグレン症候群など)、および、臓器非特異的自己免疫疾患(例えば、関節リウマチ、全身性エリテマトーデス(SLE)、円板状エリテマトーデス、多発性筋炎、強皮症、および混合結合組織病など)が挙げられる。関節リウマチ、全身性エリテマトーデス、円板状エリテマトーデス、多発性筋炎、強皮症および混合結合組織病は、膠原病として知られている。すなわち膠原病は、全身性自己免疫疾患に含まれるものである。本発明の一態様における自己免疫疾患は、膠原病であることが好ましく、全身性エリテマトーデスであることが更に好ましい。 As used herein, the term "autoimmune disease" means that an autoantibody (eg, anti-Sm antibody, anti-dsDNA antibody, rheumatoid factor (RF)) against a self-component (eg, immunoglobulin) is detected and autoimmune. Intended for diseases associated with the pathology. Autoimmune diseases include organ-specific autoimmune diseases (eg, chronic thyroiditis, primary mucosal edema, thyroid poisoning, malignant anemia, Good Pasture syndrome, acute progressive glomerulonephritis, severe myasthenia, vulgaris vulgaris). Blisters, vesicular vesicles, insulin-resistant diabetes, juvenile diabetes, Addison's disease, atrophic gastric inflammation, male infertility, premature climacteric, crystalline venous inflammation, sympathitis, polysclerosis, Ulceral colitis, primary biliary cirrhosis, chronic active hepatitis, autoimmune hemolytic anemia, paroxysmal hemochromatosis, idiopathic thrombocytopenic purpura, and Sjogren's syndrome), and organ-nonspecific self Immune diseases such as rheumatoid arthritis, systemic erythematosus (SLE), discoid erythematosus, polymyopathy, scleroderma, and mixed connective tissue disease, etc.). Rheumatoid arthritis, systemic lupus erythematosus, discoid lupus erythematosus, polymyositis, scleroderma and mixed connective tissue disease are known as collagen diseases. That is, collagen disease is included in systemic autoimmune diseases. The autoimmune disease in one embodiment of the present invention is preferably collagen disease, more preferably systemic lupus erythematosus.
DOCK8タンパク質は、DOCKファミリータンパク質のメンバーであり、グアニンヌクレオチド交換因子(guanine nucleotide exchange factor:GEF)の機能を有する分子量238kDaのタンパク質である。DOCK8タンパク質は、Rhoファミリー低分子量Gタンパク質の活性化を介してアクチン細胞骨格の重合を制御することが知られている。アクチン細胞骨格の再構成は、細胞の分化・増殖、遊走、シグナル伝達など様々な細胞機能に関わっている。 The DOCK8 protein is a member of the DOCK family protein and is a protein having a molecular weight of 238 kDa having the function of a guanine nucleotide exchange factor (GEF). The DOCK8 protein is known to regulate the polymerization of the actin cytoskeleton through activation of the Rho family small G proteins. The rearrangement of the actin cytoskeleton is involved in various cell functions such as cell differentiation / proliferation, migration, and signal transduction.
近年、ヒトDOCK8遺伝子の欠損が重症複合免疫不全症を引き起こすことが発見されたことをきっかけに、DOCK8の免疫学的な研究成果が複数報告されている(非特許文献3~8参照)。
In recent years, a number of immunological research results on DOCK8 have been reported, triggered by the discovery that a deficiency in the human DOCK8 gene causes severe combined immunodeficiency disease (see Non-Patent
なお、本明細書において、「DOCK8」は、特に説明がない場合は、DOCK8タンパク質およびDOCK8タンパク質をコードするDNA(遺伝子)の両方を意図する。 In the present specification, "DOCK8" is intended to be both a DOCK8 protein and a DNA (gene) encoding the DOCK8 protein, unless otherwise specified.
抗DOCK8抗体は、DOCK8タンパク質、または、DOCK8タンパク質の部分配列に結合するものである。このとき、結合対象であるDOCK8タンパク質の由来は、特に限定されず、例えば哺乳動物を挙げることができる。哺乳動物としては、例えば、マウス、ラット、ウサギ、ヤギ、サル、ヒトが挙げられ、好ましくは、マウス、ラット、ヒトであり、より好ましくはヒトである。 The anti-DOCK8 antibody is one that binds to the DOCK8 protein or a partial sequence of the DOCK8 protein. At this time, the origin of the DOCK8 protein to be bound is not particularly limited, and examples thereof include mammals. Examples of mammals include mice, rats, rabbits, goats, monkeys and humans, preferably mice, rats and humans, and more preferably humans.
後述する実施例では、細胞膜の表面に存在するDOCK8タンパク質が、SLEの発症メカニズムに関与していることが示唆されている。それ故に、本実施形態に係る抗DOCK8抗体は、DOCK8タンパク質の細胞膜への局在を阻害する抗体であってもよい。例えば、DOCK8タンパク質が細胞膜に局在している細胞A、および、任意の抗体が加えられた当該細胞Bの各々を、抗DOCK8抗体を用いて免疫染色し、細胞Bにおける細胞膜近傍の染色強度が、細胞Aにおける細胞膜近傍の染色強度よりも低下していれば、当該任意の抗体を、DOCK8タンパク質の細胞膜への局在を阻害する抗体であると判定することができる。 In the examples described later, it is suggested that the DOCK8 protein present on the surface of the cell membrane is involved in the pathogenic mechanism of SLE. Therefore, the anti-DOCK8 antibody according to the present embodiment may be an antibody that inhibits the localization of the DOCK8 protein to the cell membrane. For example, each of cell A in which the DOCK8 protein is localized in the cell membrane and the cell B to which an arbitrary antibody is added is immunostained with an anti-DOCK8 antibody, and the staining intensity in the vicinity of the cell membrane in the cell B is increased. If the staining intensity in the vicinity of the cell membrane in cell A is lower than that of the cell membrane, it can be determined that the arbitrary antibody is an antibody that inhibits the localization of the DOCK8 protein to the cell membrane.
より具体的に、抗DOCK8抗体は、配列番号1または5で示されるアミノ酸配列からなるヒト由来のポリペプチド、配列番号2で示されるアミノ酸配列からなるマウス由来のポリペプチド、または、これらのポリペプチドの部分配列に結合するものであってもよい。 More specifically, the anti-DOCK8 antibody is a human-derived polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 5, a mouse-derived polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, or a polypeptide thereof. It may be one that binds to a partial sequence of.
上記部分配列を構成するアミノ酸の数は、特に限定されず、例えば、400個以下、350個以下、300個以下、200個以下、100個以下、90個以下、80個以下、70個以下、60個以下、50個以下、40個以下、30個以下、20個以下、または、10個以下であってもよい。また、上記部分配列を構成するアミノ酸の数の下限値は、特に限定されず、例えば、3個、4個、5個、6個、7個、8個、9個、または、10個であってもよい。 The number of amino acids constituting the partial sequence is not particularly limited, and for example, 400 or less, 350 or less, 300 or less, 200 or less, 100 or less, 90 or less, 80 or less, 70 or less, It may be 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, or 10 or less. The lower limit of the number of amino acids constituting the partial sequence is not particularly limited, and is, for example, 3, 4, 5, 6, 7, 8, 9, or 10. You may.
抗DOCK8抗体は、DOCK8タンパク質、または、DOCK8タンパク質の部分配列に特異的に結合する抗体である。なお、本明細書において、抗体がDOCK8タンパク質、または、DOCK8タンパク質の部分配列に「特異的に結合する」とは、その抗体が、他のポリペプチドに対してよりも、DOCK8タンパク質、または、DOCK8タンパク質の部分配列に対して、高い親和性で結合することを意図する。ここで「高い親和性」とは、例えば、結合定数(Ka:binding constant)が、107M-1以上、好ましくは108M-1以上、より好ましくは109M-1以上、より好ましくは1010M-1以上、より好ましくは1011M-1以上、より好ましくは1012M-1以上、最も好ましくは1013M-1以上である親和性を意図する。The anti-DOCK8 antibody is a DOCK8 protein or an antibody that specifically binds to a partial sequence of the DOCK8 protein. It should be noted that, in the present specification, an antibody "specifically binds" to a DOCK8 protein or a partial sequence of a DOCK8 protein means that the antibody has a DOCK8 protein or DOCK8 rather than to other polypeptides. It is intended to bind with high affinity to partial sequences of proteins. Here, "high affinity" means, for example, that the binding constant (Ka: binding constant) is 10 7 M -1 or more, preferably 10 8 M -1 or more, more preferably 10 9 M -1 or more, and more preferably. Is intended for affinity of 10 10 M -1 or higher, more preferably 10 11 M -1 or higher, more preferably 10 12 M -1 or higher, and most preferably 10 13 M -1 or higher.
抗DOCK8抗体は、抗DOCK8抗体の全体を含むもの、抗DOCK8抗体の全体からなるもの、抗DOCK8抗体の断片を含むもの、または、抗DOCK8抗体の断片からなるものであってもよい。例えば、DOCK8抗体は、ポリクローナル抗体、モノクローナル抗体、キメラ抗体、ヒト化抗体、ヒト抗体、または、これらの断片(例えば、F(ab’)2、Fab’、Fab、または、Fv)であってもよい。例えば、抗DOCK8抗体がモノクローナル抗体であれば、抗DOCK8抗体がDOCK8タンパク質以外の対象に非特異的に結合することを低減することができるので、本実施の形態の自己免疫疾患の予防および/または治療剤の薬理効果を上げることができるとともに、本実施の形態の自己免疫疾患の予防および/または治療剤の副作用の発生を抑えることができる。抗DOCK8抗体は、IgA、IgD、IgE、IgG、IgM、または、これらの断片であってもよい。The anti-DOCK8 antibody may be an entire anti-DOCK8 antibody, an entire anti-DOCK8 antibody, an anti-DOCK8 antibody fragment, or an anti-DOCK8 antibody fragment. For example, the DOCK8 antibody may be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, or a fragment thereof (for example, F (ab') 2 , Fab', Fab, or Fv). good. For example, if the anti-DOCK8 antibody is a monoclonal antibody, it is possible to reduce the non-specific binding of the anti-DOCK8 antibody to a target other than the DOCK8 protein, and thus the prevention and / or prevention of autoimmune diseases of the present embodiment. The pharmacological effect of the therapeutic agent can be enhanced, and the prevention of the autoimmune disease of the present embodiment and / or the occurrence of side effects of the therapeutic agent can be suppressed. The anti-DOCK8 antibody may be IgA, IgD, IgE, IgG, IgM, or a fragment thereof.
抗DOCK8抗体は、周知の方法(例えば、(1)HarLowら、「Antibodies:A laboratory manual,Cold Spring Harbor Laboratory,New York(1988)」、および、(2)岩崎ら、「単クローン抗体 ハイブリドーマとELISA、講談社(1991)」)にしたがって作製することができる。勿論、抗DOCK8抗体として、市販の抗体を用いることも可能である。 Anti-DOCK8 antibodies are described by well-known methods (eg, (1) HarLow et al., "Antibodies: A laboratory manual, Cold Spring Harbor Laboratory, New York (1988)", and (2) Iwasaki et al., "Single clone antibody hybridoma". It can be produced according to ELISA, Kodansha (1991) "). Of course, it is also possible to use a commercially available antibody as the anti-DOCK8 antibody.
モノクローナル抗体は、当該分野において周知の方法(例えば、(1)ハイブリドーマ法(Kohler,G.およびMilstein,C.,Nature 256,495-497(1975))、(2)トリオーマ法、(3)ヒトB細胞ハイブリドーマ法(Kozbor,Immunology Today 4,72(1983))、および、(4)EBV-ハイブリドーマ法(Monoclonal Antibodies and Cancer Therapy,Alan R Liss,Inc.,77-96(1985)))にしたがって作製することができる。
Monoclonal antibodies are used by methods well known in the art (eg, (1) hybridoma method (Kohler, G. and Milstein, C., Nature 256, 495-497 (1975)), (2) trioma method, (3) human. According to the B-cell hybridoma method (Kozbor,
モノクローナル抗体は、ファージディスプレイ法にしたがって作製することもできる。ファージディスプレイ法によるモノクローナル抗体の作製方法は、公知の方法を用いればよい。ファージディスプレイ法によるモノクローナル抗体の作製方法の一例を、以下に説明する。まず、DOCK8タンパク質を免疫した動物からmRNAを調製し、当該mRNAを鋳型としてcDNAを調製し、当該cDNAから、抗体の可変領域のみをコードする1本鎖抗体(scFV)遺伝子を作製する。次いで、当該1本鎖抗体遺伝子をファージミドベクターに挿入し、当該ファージミドベクターを大腸菌に導入した後、当該大腸菌にファージを感染させる。これによって、scFV抗体をファージ被膜上に発現させることができる。ファージ被膜上に発現されたscFV抗体の中から、DOCK8タンパク質に結合するものをスクリーニングすることによって、抗DOCK8モノクローナル抗体を作製することができる。なお、mRNAの調製、cDNAの調製、1本鎖抗体遺伝子のファージミドベクターへの挿入、ファージミドベクターの大腸菌への導入、ファージの感染、および、抗体のスクリーニングは、周知の方法にしたがって行えばよい。 Monoclonal antibodies can also be produced according to the phage display method. As a method for producing a monoclonal antibody by the phage display method, a known method may be used. An example of a method for producing a monoclonal antibody by the phage display method will be described below. First, mRNA is prepared from an animal immunized with the DOCK8 protein, cDNA is prepared using the mRNA as a template, and a single-chain antibody (scFV) gene encoding only the variable region of the antibody is prepared from the cDNA. Next, the single-stranded antibody gene is inserted into a phagemid vector, the phagemid vector is introduced into Escherichia coli, and then the E. coli is infected with the phage. This allows the scFV antibody to be expressed on the phage capsule. An anti-DOCK8 monoclonal antibody can be prepared by screening an scFV antibody expressed on the phage capsule that binds to the DOCK8 protein. Preparation of mRNA, preparation of cDNA, insertion of a single-stranded antibody gene into a phagemid vector, introduction of a phagemid vector into Escherichia coli, infection of a phage, and screening of an antibody may be carried out according to a well-known method.
キメラ抗体とは、定常領域がヒトの抗体の定常領域に置き換えられている抗体を意図する。キメラ抗体は、周知方法にしたがって作製され得る(例えば、欧州特許公開公報EP0125023を参照)。 Chimeric antibodies are intended as antibodies in which the constant region is replaced by the constant region of a human antibody. Chimeric antibodies can be made according to well-known methods (see, eg, European Patent Publication EP0125023).
ヒト化抗体とは、H鎖およびL鎖の相補性決定領域(CDR:complementarity determining region)以外がヒトの抗体の構造に置き換えられている抗体を意図する。 The humanized antibody is intended to be an antibody in which the structure of a human antibody is replaced by other than the complementarity determining regions (CDRs) of the H chain and the L chain.
ヒト抗体とは、ヒトの抗体生産に関与する遺伝子が導入されたトランスジェニック動物を用いて作製された抗体を意図する(例えば、欧州特許公開公報EP0546073を参照)。 The human antibody is intended to be an antibody produced using a transgenic animal into which a gene involved in human antibody production has been introduced (see, for example, EP0546073).
F(ab’)2、Fab’、Fab、および、Fvは、抗体の全体をプロテアーゼ(例えば、パパイン、または、ペプシン)によって分解し、必要に応じて、分解物を更に還元することによって得ることができる。また、F(ab’)2、Fab’、Fab、および、Fvは、抗体を生産するハイブリドーマから、これらのcDNAを単離し、当該cDNAを発現ベクターに挿入し、当該発現ベクターを宿主に導入することによって得ることができる。この場合には、抗体フラグメントは、抗体フラグメントと別のタンパク質との融合タンパク質として得ることもできる。F (ab') 2 , Fab', Fab, and Fv can be obtained by degrading the entire antibody with a protease (eg, papain or pepsin) and, if necessary, further reducing the degradation product. Can be done. In addition, F (ab') 2 , Fab', Fab, and Fv isolate these cDNAs from hybridomas that produce antibodies, insert the cDNAs into expression vectors, and introduce the expression vectors into the host. Can be obtained by In this case, the antibody fragment can also be obtained as a fusion protein of the antibody fragment and another protein.
本実施の形態の自己免疫疾患の予防および/または治療剤に含有されている抗DOCK8抗体の量は、特に限定されず、例えば、予防および/または治療剤を100重量%とした場合に、0.001重量%~100重量%であってもよく、0.01重量%~100重量%であってもよく、0.1重量%~100重量%であってもよく、0.1重量%~95重量%であってもよく、0.1重量%~90重量%であってもよく、0.1重量%~80重量%であってもよく、0.1重量%~70重量%であってもよく、0.1重量%~60重量%であってもよく、0.1重量%~50重量%であってもよく、0.1重量%~40重量%であってもよく、0.1重量%~30重量%であってもよく、0.1重量%~20重量%であってもよく、0.1重量%~10重量%であってもよい。 The amount of the anti-DOCK8 antibody contained in the prophylactic and / or therapeutic agent for autoimmune disease of the present embodiment is not particularly limited, and is, for example, 0 when the prophylactic and / or therapeutic agent is 100% by weight. .001% by weight to 100% by weight, 0.01% by weight to 100% by weight, 0.1% by weight to 100% by weight, 0.1% by weight to It may be 95% by weight, 0.1% by weight to 90% by weight, 0.1% by weight to 80% by weight, 0.1% by weight to 70% by weight. It may be 0.1% by weight to 60% by weight, 0.1% by weight to 50% by weight, 0.1% by weight to 40% by weight, 0. It may be 1% by weight to 30% by weight, 0.1% by weight to 20% by weight, or 0.1% by weight to 10% by weight.
本実施の形態の自己免疫疾患の予防および/または治療剤に含有されている抗DOCK8抗体の量は、特に限定されず、例えば、0.01mg~1000mg、0.1mg~1000mg、1mg~1000mg、1mg~900mg、1mg~800mg、1mg~700mg、1mg~600mg、1mg~500mg、1mg~400mg、1mg~300mg、1mg~200mg、または、1mg~100mgであってもよい。 The amount of the anti-DOCK8 antibody contained in the prophylactic and / or therapeutic agent for the autoimmune disease of the present embodiment is not particularly limited, and is, for example, 0.01 mg to 1000 mg, 0.1 mg to 1000 mg, 1 mg to 1000 mg, and the like. It may be 1 mg to 900 mg, 1 mg to 800 mg, 1 mg to 700 mg, 1 mg to 600 mg, 1 mg to 500 mg, 1 mg to 400 mg, 1 mg to 300 mg, 1 mg to 200 mg, or 1 mg to 100 mg.
本実施の形態の自己免疫疾患の予防および/または治療剤は、抗DOCK8抗体以外の成分(例えば、薬学的に受容可能なキャリア)を含んでいてもよい。抗DOCK8抗体以外の成分としては、本実施の形態の自己免疫疾患の予防および/または治療剤が固形製剤である場合には、賦形剤、滑沢剤、結合剤および崩壊剤を挙げることができ、本実施の形態の自己免疫疾患の予防および/または治療剤が液状製剤である場合には、溶剤、溶解補助剤、懸濁剤、等張化剤、緩衝剤および無痛化剤を挙げることができる。その他、抗DOCK8抗体以外の成分として、防腐剤、抗酸化剤および安定化剤も挙げることができる。 The prophylactic and / or therapeutic agent for autoimmune diseases of this embodiment may contain components other than the anti-DOCK8 antibody (eg, pharmaceutically acceptable carriers). Examples of the component other than the anti-DOCK8 antibody include excipients, lubricants, binders and disintegrants when the prophylactic and / or therapeutic agent for the autoimmune disease of the present embodiment is a solid preparation. When the preventive and / or therapeutic agent for the autoimmune disease of the present embodiment is a liquid preparation, the solvent, the solubilizing agent, the suspending agent, the tonicity agent, the buffering agent and the soothing agent can be mentioned. Can be done. In addition, as components other than the anti-DOCK8 antibody, preservatives, antioxidants and stabilizers can also be mentioned.
上記「賦形剤」としては、例えば、乳糖、白糖、D-マンニトール、キシリトール、ソルビトール、エリスリトール、デンプンおよび結晶セルロースを挙げることが、これらに限定されない。 Examples of the above-mentioned "excipient" include, but are not limited to, lactose, sucrose, D-mannitol, xylitol, sorbitol, erythritol, starch and crystalline cellulose.
上記「滑沢剤」としては、例えば、ステアリン酸マグネシウム、ステアリン酸カルシウム、ワックス、タルクおよびコロイドシリカを挙げることが、これらに限定されない。 Examples of the above-mentioned "lubricating agent" include, but are not limited to, magnesium stearate, calcium stearate, wax, talc and colloidal silica.
上記「結合剤」としては、例えば、α化デンプン、メチルセルロース、結晶セルロース、白糖、D-マンニトール、トレハロース、デキストリン、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロースおよびポリビニルピロリドンを挙げることが、これらに限定されない。 Examples of the above-mentioned "binder" include, but are not limited to, pregelatinized starch, methyl cellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, hydroxypropyl cellulose, hydroxypropyl methyl cellulose and polyvinylpyrrolidone.
上記「崩壊剤」としては、例えば、デンプン、カルボキシメチルセルロース、低置換度ヒドロキシプロピルセルロース、カルボキシメチルセルロースカルシウム、クロスカルメロースナトリウムおよびカルボキシメチルスターチナトリウムを挙げることが、これらに限定されない。 Examples of the above-mentioned "disintegrant" include, but are not limited to, starch, carboxymethyl cellulose, low-degree-of-substitution hydroxypropyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium and carboxymethyl starch sodium.
上記「溶剤」としては、例えば、注射用水、アルコール、プロピレングリコール、マクロゴール、ゴマ油、トウモロコシ油およびトリカプリリンを挙げることが、これらに限定されない。 Examples of the "solvent" include, but are not limited to, water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil and tricaprylin.
上記「溶解補助剤」としては、例えば、ポリエチレングリコール、プロピレングリコール、D-マンニトール、トレハロース、安息香酸ベンジル、エタノール、トリスアミノメタン、コレステロール、トリエタノールアミン、炭酸ナトリウムおよびクエン酸ナトリウムを挙げることが、これらに限定されない。 Examples of the above-mentioned "solubilizing agent" include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate and sodium citrate. Not limited to these.
上記「懸濁剤」としては、例えば、界面活性剤(例えば、ステアリルトリエタノールアミン、ラウリル硫酸ナトリウム、ラウリルアミノプロピオン酸、レシチン、塩化ベンザルコニウム、塩化ベンゼトニウム、モノステアリン酸グリセリン)および親水性高分子(例えば、ポリビニルアルコール、ポリビニルピロリドン、カルボキシメチルセルロースナトリウム、メチルセルロース、ヒドロキシメチルセルロース、ヒドロキシエチルセルロース、ヒドロキシプロピルセルロース)を挙げることが、これらに限定されない。 Examples of the above-mentioned "suspending agent" include surfactants (for example, stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate) and highly hydrophilic. Molecules (eg, polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose) are not limited thereto.
上記「等張化剤」としては、例えば、塩化ナトリウム、グリセリンおよびD-マンニトールを挙げることが、これらに限定されない。 Examples of the above-mentioned "isotonic agent" include, but are not limited to, sodium chloride, glycerin and D-mannitol.
上記「緩衝剤」としては、例えば、リン酸塩、酢酸塩、炭酸塩およびクエン酸塩を挙げることが、これらに限定されない。 Examples of the above-mentioned "buffering agent" include, but are not limited to, phosphates, acetates, carbonates and citrates.
上記「無痛化剤」としては、例えば、ベンジルアルコールを挙げることが、これに限定されない。 Examples of the above-mentioned "painless agent" include, but are not limited to, benzyl alcohol.
上記「防腐剤」としては、例えば、パラオキシ安息香酸エステル類、クロロブタノール、ベンジルアルコール、フェネチルアルコール、デヒドロ酢酸およびソルビン酸を挙げることが、これらに限定されない。 Examples of the above-mentioned "preservative" include, but are not limited to, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid and sorbic acid.
上記「抗酸化剤」としては、例えば、亜硫酸塩およびアスコルビン酸を挙げることが、これらに限定されない。 Examples of the above-mentioned "antioxidant" include, but are not limited to, sulfites and ascorbic acid.
上記「安定化剤」としては、製薬分野において通常用いられるものであればよく、特に限定されない。 The above-mentioned "stabilizer" may be any one usually used in the pharmaceutical field and is not particularly limited.
本実施の形態の自己免疫疾患の予防および/または治療剤に含有されている抗DOCK8抗体以外の成分の量は、特に限定されず、例えば、予防および/または治療剤を100重量%とした場合に、0重量%~99.999重量%であってもよく、0重量%~99.99重量%であってもよく、0重量%~99.9重量%であってもよく、5重量%~99.9重量%であってもよく、10重量%~99.9重量%であってもよく、20重量%~99.9重量%であってもよく、30重量%~99.9重量%であってもよく、40重量%~99.9重量%であってもよく、50重量%~99.9重量%であってもよく、60重量%~99.9重量%であってもよく、70重量%~99.9重量%であってもよく、80重量%~99.9重量%であってもよく、90重量%~99.9重量%であってもよい。 The amount of components other than the anti-DOCK8 antibody contained in the prophylactic and / or therapeutic agent for autoimmune diseases of the present embodiment is not particularly limited, and for example, when the prophylactic and / or therapeutic agent is 100% by weight. In addition, it may be 0% by weight to 99.99% by weight, 0% by weight to 99.99% by weight, 0% by weight to 99.9% by weight, or 5% by weight. It may be up to 99.9% by weight, 10% by weight to 99.9% by weight, 20% by weight to 99.9% by weight, and 30% by weight to 99.9% by weight. %, 40% by weight to 99.9% by weight, 50% by weight to 99.9% by weight, 60% by weight to 99.9% by weight. It may be 70% by weight to 99.9% by weight, 80% by weight to 99.9% by weight, or 90% by weight to 99.9% by weight.
本実施の形態の自己免疫疾患の予防および/または治療剤に含有されている抗DOCK8抗体以外の成分の量は、特に限定されず、例えば、0.01mg~1000mg、0.1mg~1000mg、1mg~1000mg、1mg~900mg、1mg~800mg、1mg~700mg、1mg~600mg、1mg~500mg、1mg~400mg、1mg~300mg、1mg~200mg、または、1mg~100mgであってもよい。 The amount of components other than the anti-DOCK8 antibody contained in the preventive and / or therapeutic agent for autoimmune diseases of the present embodiment is not particularly limited, and is, for example, 0.01 mg to 1000 mg, 0.1 mg to 1000 mg, and 1 mg. It may be ~ 1000 mg, 1 mg to 900 mg, 1 mg to 800 mg, 1 mg to 700 mg, 1 mg to 600 mg, 1 mg to 500 mg, 1 mg to 400 mg, 1 mg to 300 mg, 1 mg to 200 mg, or 1 mg to 100 mg.
本実施の形態の自己免疫疾患の予防および/または治療剤の投与方法は、特に限定されず、経口的に投与されてもよいし、非経口的に、静脈内、直腸内、腹腔内、筋肉内、鼻内または皮下に投与されてもよい。 The method for administering the prophylactic and / or therapeutic agent for the autoimmune disease of the present embodiment is not particularly limited and may be orally administered, or parenterally, intravenously, intrarectally, intraperitoneally, or muscle. It may be administered intranasally, intranasally or subcutaneously.
本実施の形態の自己免疫疾患の予防および/または治療剤の剤形は、特に限定されず、例えば、注射剤(例えば、腹腔内注射、静脈注射剤、皮下注射剤、皮内注射剤、筋肉注射剤、または、点滴注射剤)、点鼻剤、または、坐剤であってもよい。 The dosage form of the preventive and / or therapeutic agent for autoimmune disease of the present embodiment is not particularly limited, and is, for example, an injection (for example, intraperitoneal injection, intravenous injection, subcutaneous injection, intradermal injection, muscle). It may be an injection (or a drip injection), a nasal drop, or a suppository.
〔2.自己免疫疾患の予防用および/または治療用のワクチン〕
後述する実施例に示すように、抗DOCK8抗体によって、自己免疫疾患を予防および/または治療することができる。このことは、生体に投与されて抗DOCK8抗体の生産を誘導することができる物質(例えば、抗原)を含有しているワクチンも、自己免疫疾患を予防および/または治療することができることを示している。[2. Vaccines for the prevention and / or treatment of autoimmune diseases]
As shown in the examples described below, anti-DOCK8 antibodies can prevent and / or treat autoimmune diseases. This indicates that vaccines containing substances (eg, antigens) that can be administered to the body to induce the production of anti-DOCK8 antibodies can also prevent and / or treat autoimmune diseases. There is.
それ故に、本実施の形態の自己免疫疾患の予防用および/または治療用のワクチンは、DOCK8タンパク質、DOCK8タンパク質の部分配列、または、DOCK8タンパク質若しくはDOCK8タンパク質の部分配列の発現ベクターを有効成分として含有している。 Therefore, the vaccine for the prevention and / or treatment of autoimmune diseases of the present embodiment contains the expression vector of the DOCK8 protein, the partial sequence of the DOCK8 protein, or the partial sequence of the DOCK8 protein or the DOCK8 protein as an active ingredient. is doing.
例えば、本実施の形態の自己免疫疾患の予防用および/または治療用のワクチンは、投与対象とは異なる種に由来するDOCK8タンパク質、投与対象とは異なる種に由来するDOCK8タンパク質の部分配列、または、投与対象とは異なる種に由来するDOCK8タンパク質若しくはDOCK8タンパク質の部分配列の発現ベクターを有効成分として含有していてもよい。 For example, the vaccine for the prevention and / or treatment of an autoimmune disease of the present embodiment may be a DOCK8 protein derived from a species different from the subject, a partial sequence of the DOCK8 protein derived from a species different from the subject, or , A DOCK8 protein derived from a species different from the administration target or an expression vector of a partial sequence of the DOCK8 protein may be contained as an active ingredient.
本実施の形態の自己免疫疾患の予防用および/または治療用のワクチンと、上述した自己免疫疾患の予防および/または治療剤とは、有効成分として用いる物質のみが異なり、その他の構成は同じであってもよい。本実施の形態の自己免疫疾患の予防用および/または治療用のワクチンに関して、有効成分以外については既に説明したので、ここでは、その説明を省略する。 The vaccine for the prevention and / or treatment of autoimmune diseases of the present embodiment and the above-mentioned prophylactic and / or therapeutic agent for autoimmune diseases differ only in the substance used as an active ingredient, and have the same other configurations. There may be. Since the vaccines for the prevention and / or the treatment of autoimmune diseases of the present embodiment have already been described except for the active ingredient, the description thereof will be omitted here.
有効成分であるDOCK8タンパク質、および、DOCK8タンパク質の部分配列としては、配列番号1または5で示されるアミノ酸配列からなるヒト由来のポリペプチド、配列番号2で示されるアミノ酸配列からなるマウス由来のポリペプチド、および、これらのポリペプチドの部分配列を挙げることができる。 The DOCK8 protein, which is an active ingredient, and the partial sequence of the DOCK8 protein include a human-derived polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 5, and a mouse-derived polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2. , And partial sequences of these polypeptides.
上記部分配列を構成するアミノ酸の数は、特に限定されず、例えば、400個以下、350個以下、300個以下、200個以下、100個以下、90個以下、80個以下、70個以下、60個以下、50個以下、40個以下、30個以下、20個以下、または、10個以下であってもよい。また、上記部分配列は、DOCK8タンパク質の中の部分配列であればよく、DOCK8タンパク質中の位置は限定されない。 The number of amino acids constituting the partial sequence is not particularly limited, and for example, 400 or less, 350 or less, 300 or less, 200 or less, 100 or less, 90 or less, 80 or less, 70 or less, It may be 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, or 10 or less. Further, the partial sequence may be a partial sequence in the DOCK8 protein, and the position in the DOCK8 protein is not limited.
DOCK8タンパク質、および、DOCK8タンパク質の部分配列は、公知の方法によって作製することができる。例えば、(i)公知のペプチド合成装置を用いて作製することも可能であるし、(ii)DOCK8タンパク質、および、DOCK8タンパク質の部分配列をコードするcDNAを発現ベクターに挿入し、当該発現ベクターを所望の宿主(例えば、大腸菌、および、酵母など)に導入することによって作製することも可能である。 The DOCK8 protein and the partial sequence of the DOCK8 protein can be prepared by a known method. For example, (i) it can be prepared using a known peptide synthesizer, or (ii) a DOCK8 protein and a cDNA encoding a partial sequence of the DOCK8 protein are inserted into an expression vector, and the expression vector is inserted. It can also be produced by introduction into a desired host (eg, E. coli, yeast, etc.).
有効成分であるDOCK8タンパク質、または、DOCK8タンパク質の部分配列の発現ベクターは、DOCK8タンパク質、または、DOCK8タンパク質の部分配列をコードするcDNAが、ベクターに発現可能に挿入されているものである。 The expression vector of the DOCK8 protein or the partial sequence of the DOCK8 protein as the active ingredient is one in which the cDNA encoding the DOCK8 protein or the partial sequence of the DOCK8 protein is expressively inserted into the vector.
ベクターの具体的な例としては、特に限定されず、例えば、プラスミド、ウイルスベクター(例えば、ワクシニアウイルスベクター、ヘルペスウイルスベクター、アデノウイルスベクター、アデノ随伴ウイルスベクター、および、レトロウイスルベクター)、または、ウイルス粒子を挙げることができる。当該ベクターに挿入されるプロモーターの具体的な例としては、特に限定されず、上述したベクターに応じて適宜選択すればよい。 Specific examples of the vector are not particularly limited, and are, for example, a plasmid, a viral vector (for example, a vaccinia virus vector, a herpes virus vector, an adenovirus vector, an adeno-associated virus vector, and a retrovirus vector), or a virus. Particles can be mentioned. The specific example of the promoter inserted into the vector is not particularly limited, and may be appropriately selected depending on the above-mentioned vector.
上記ベクターの中に、DOCK8タンパク質、または、DOCK8タンパク質の部分配列をコードするcDNAが、発現可能に挿入される。当該cDNAの具体的な塩基配列は、特に限定されない。DOCK8タンパク質、または、DOCK8タンパク質の部分配列をコードするcDNAとしては、配列番号3で示される塩基配列からなるヒト由来のポリヌクレオチド、配列番号4で示される塩基配列からなるマウス由来のポリヌクレオチド、および、これらのポリヌクレオチドの部分配列を挙げることができる。 A DOCK8 protein or a cDNA encoding a partial sequence of the DOCK8 protein is inserted into the vector in an expressible manner. The specific base sequence of the cDNA is not particularly limited. The cDNA encoding the DOCK8 protein or the partial sequence of the DOCK8 protein includes a human-derived polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 3, a mouse-derived polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 4, and a polynucleotide derived from a mouse. , Partial sequences of these polynucleotides can be mentioned.
本実施の形態の自己免疫疾患の予防用および/または治療用のワクチンに含有されている有効成分の量は、特に限定されず、例えば、予防用および/または治療用のワクチンを100重量%とした場合に、0.001重量%~100重量%であってもよく、0.01重量%~100重量%であってもよく、0.1重量%~100重量%であってもよく、0.1重量%~95重量%であってもよく、0.1重量%~90重量%であってもよく、0.1重量%~80重量%であってもよく、0.1重量%~70重量%であってもよく、0.1重量%~60重量%であってもよく、0.1重量%~50重量%であってもよく、0.1重量%~40重量%であってもよく、0.1重量%~30重量%であってもよく、0.1重量%~20重量%であってもよく、0.1重量%~10重量%であってもよい。 The amount of the active ingredient contained in the preventive and / or therapeutic vaccine of the present embodiment is not particularly limited, and for example, the preventive and / or therapeutic vaccine is 100% by weight. In this case, it may be 0.001% by weight to 100% by weight, 0.01% by weight to 100% by weight, 0.1% by weight to 100% by weight, or 0. .1% by weight to 95% by weight, 0.1% by weight to 90% by weight, 0.1% by weight to 80% by weight, 0.1% by weight to It may be 70% by weight, 0.1% by weight to 60% by weight, 0.1% by weight to 50% by weight, 0.1% by weight to 40% by weight. It may be 0.1% by weight to 30% by weight, 0.1% by weight to 20% by weight, or 0.1% by weight to 10% by weight.
本実施の形態の自己免疫疾患の予防用および/または治療用のワクチンに含有されている抗有効成分の量は、特に限定されず、例えば、0.01mg~1000mg、0.1mg~1000mg、1mg~1000mg、1mg~900mg、1mg~800mg、1mg~700mg、1mg~600mg、1mg~500mg、1mg~400mg、1mg~300mg、1mg~200mg、または、1mg~100mgであってもよい。 The amount of the anti-active ingredient contained in the vaccine for the prevention and / or treatment of the autoimmune disease of the present embodiment is not particularly limited, and is, for example, 0.01 mg to 1000 mg, 0.1 mg to 1000 mg, 1 mg. It may be ~ 1000 mg, 1 mg to 900 mg, 1 mg to 800 mg, 1 mg to 700 mg, 1 mg to 600 mg, 1 mg to 500 mg, 1 mg to 400 mg, 1 mg to 300 mg, 1 mg to 200 mg, or 1 mg to 100 mg.
本発明の一態様は、以下のように構成することもできる。 One aspect of the present invention can also be configured as follows.
本発明の一態様に係る自己免疫疾患の予防および/または治療剤は、抗DOCK8抗体を有効成分として含有することを特徴としている。 The prophylactic and / or therapeutic agent for an autoimmune disease according to one aspect of the present invention is characterized by containing an anti-DOCK8 antibody as an active ingredient.
本発明の一態様に係る自己免疫疾患の予防および/または治療剤では、上記自己免疫疾患は、全身性エリテマトーデス(SLE)であることが好ましい。 In the prophylactic and / or therapeutic agent for an autoimmune disease according to one aspect of the present invention, the autoimmune disease is preferably systemic lupus erythematosus (SLE).
本発明の一態様に係る自己免疫疾患の予防および/または治療剤では、上記抗DOCK8抗体は、配列番号1、2または5で示されるアミノ酸配列からなるポリペプチド、または、当該ポリペプチドの部分配列に結合するものであることが好ましい。 In the prophylactic and / or therapeutic agent for autoimmune diseases according to one aspect of the present invention, the anti-DOCK8 antibody is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1, 2 or 5, or a partial sequence of the polypeptide. It is preferable that it binds to.
本発明の一態様に係る自己免疫疾患の予防および/または治療剤では、上記抗DOCK8抗体は、モノクローナル抗体であることが好ましい。 In the prophylactic and / or therapeutic agent for autoimmune diseases according to one aspect of the present invention, the anti-DOCK8 antibody is preferably a monoclonal antibody.
本発明の一態様に係る自己免疫疾患の予防用および/または治療用のワクチンは、DOCK8タンパク質、DOCK8タンパク質の部分配列、または、DOCK8タンパク質若しくはDOCK8タンパク質の部分配列の発現ベクターを有効成分として含有していることを特徴としている。 The vaccine for the prevention and / or treatment of an autoimmune disease according to one aspect of the present invention contains an expression vector of a DOCK8 protein, a partial sequence of the DOCK8 protein, or a partial sequence of the DOCK8 protein or the DOCK8 protein as an active ingredient. It is characterized by being.
〔試験1〕
BALB/cマウス(Charles River Japan Inc.)に、卵白アルブミン(OVA:grade V; Sigma)0.5mg、または、PBSを、5日毎に繰り返し腹腔内投与した。これによって、自己免疫疾患の症状を呈するマウスを作製した。[Test 1]
BALB / c mice (Charles River Japan Inc.) were repeatedly intraperitoneally administered with 0.5 mg of ovalbumin (OVA: grade V; Sigma) or PBS every 5 days. As a result, mice exhibiting symptoms of autoimmune disease were produced.
6回目、9回目および12回目のOVAの投与、並びに、6回目、9回目および12回目のPBSの投与の24時間前に、rabbit由来の抗DOCK8抗体50μg、または、control IgG(rabbit IgG)50μgを、BALB/cマウスの腹腔内へ投与した。 24 hours before the 6th, 9th and 12th OVA administrations and the 6th, 9th and 12th PBS administrations, 50 μg of anti-DOCK8 antibody derived from rabbit or 50 μg of control IgG (rabbit IgG). Was intraperitoneally administered to BALB / c mice.
なお、当該抗DOCK8抗体としては、市販の抗DOCK8ポリクローナル抗体(proteintech社、DOCK8 Polyclonal Antibody、Catalog No. 11622-1-AP、抗原:配列番号5で示されるアミノ酸配列からなるポリペプチド)を用いた。 As the anti-DOCK8 antibody, a commercially available anti-DOCK8 polyclonal antibody (proteintech, DOCK8 Polyclonal Antibody, Catalog No. 11622-1-AP, antigen: polypeptide consisting of the amino acid sequence shown by SEQ ID NO: 5) was used. ..
12回目のOVAの投与後、および、12回目のPBSの投与後に、アルブスティックス(登録商標)(SIEMENS)を用いた呈色反応により、全身性エリテマトーデスでの腎炎発症の指標となるタンパク尿(proteinuria)を検出した。また、周知の方法にしたがってHE(Hematoxylin-Eosin)染色を行い、WHOの分類にしたがって腎臓の病理学的評価を行った。具体的には、「Weening JJ et al., The classification of glomerulonephritis in systemic lupus erythematosus revised. J Am Soc Nephrol 15(2):241-250, 2004」に記載の基準にしたがって、観察した腎臓の症状を、クラスI~II、クラスIII、クラスIV、および、クラスVに分類し、腎臓の病理学的評価を行った。 After the 12th OVA administration and after the 12th PBS administration, proteinuria (proteinuria), which is an indicator of the onset of nephritis in systemic lupus erythematosus, by a color reaction using Arbusticus (registered trademark) (SIEMENS). proteinuria) was detected. In addition, HE (Hematoxylin-Eosin) staining was performed according to a well-known method, and pathological evaluation of the kidney was performed according to the WHO classification. Specifically, the renal symptoms observed according to the criteria described in "Weening JJ et al., The classification of glomerulonephritis in systemic lupus erythematosus revised. J Am Soc Nephrol 15 (2): 241-250, 2004" , Class I-II, Class III, Class IV, and Class V, and pathological evaluation of the kidney was performed.
図1に、タンパク尿の試験結果を示し、下記の表1に、WHOの分類にしたがった腎臓の病理学的評価を示す。 FIG. 1 shows the test results of proteinuria, and Table 1 below shows the pathological evaluation of the kidney according to the WHO classification.
図1に示すように、6回目、9回目および12回目のOVAの投与前にcontrol IgGを投与したBALB/cマウス群(図1の「OVA×12 + control IgG」)では、タンパク尿が検出された。一方、6回目、9回目および12回目のOVAの投与前に抗DOCK8抗体を投与したBALB/cマウス群(図1の「OVA×12 + anti-DOCK8 Ab」)では、タンパク尿のレベルが低下した。なお、図1において「PBS×12 + control IgG」は、6回目、9回目および12回目のPBSの投与前にcontrol IgGを投与したBALB/cマウス群の試験結果である。 As shown in FIG. 1, proteinuria was detected in the BALB / c mouse group (“OVA × 12 + control IgG” in FIG. 1) to which control IgG was administered before the 6th, 9th, and 12th OVA administrations. Was done. On the other hand, in the BALB / c mouse group (“OVA × 12 + anti-DOCK8 Ab” in FIG. 1) to which the anti-DOCK8 antibody was administered before the 6th, 9th and 12th OVA administrations, the level of proteinuria decreased. did. In FIG. 1, "PBS x 12 + control IgG" is a test result of a BALB / c mouse group to which control IgG was administered before the 6th, 9th, and 12th administration of PBS.
また、表1に示すように、6回目、9回目および12回目のOVAの投与前にcontrol IgGを投与したBALB/cマウス群(表1の「OVA×12 + control IgG」)では、WHO分類におけるIV型およびV型など重症の腎炎が検出された。一方、6回目、9回目および12回目のOVAの投与前に抗DOCK8抗体を投与したBALB/cマウス群(表1の「OVA×12 + anti-DOCK8 Ab」)では、腎炎がほぼ完全に抑制された。なお、表1において「PBS×12 + control IgG」は、6回目、9回目および12回目のPBSの投与前にcontrol IgGを投与したBALB/cマウス群の試験結果である。 In addition, as shown in Table 1, the BALB / c mouse group (“OVA × 12 + control IgG” in Table 1) to which control IgG was administered before the 6th, 9th, and 12th OVA administrations was classified as WHO. Severe nephritis such as type IV and type V was detected in. On the other hand, in the BALB / c mouse group (“OVA × 12 + anti-DOCK8 Ab” in Table 1) to which the anti-DOCK8 antibody was administered before the 6th, 9th and 12th OVA administrations, nephritis was almost completely suppressed. Was done. In Table 1, "PBS x 12 + control IgG" is the test result of the BALB / c mouse group to which control IgG was administered before the 6th, 9th and 12th administration of PBS.
BALB/cマウス(Charles River Japan Inc.)に、卵白アルブミン(OVA:grade V; Sigma)0.5mgを、5日毎に繰り返し腹腔内投与した。これによって、自己免疫疾患の症状を呈するマウスを作製した。
BALB / c mice (Charles River Japan Inc.) were repeatedly intraperitoneally administered 0.5 mg of ovalbumin (OVA: grade V; Sigma) every 5 days. As a result, mice exhibiting symptoms of autoimmune disease were produced.
6回目、9回目および12回目のOVAの投与の24時間前に、rabbit由来の抗DOCK8抗体(〔試験1〕に用いたものと同じ抗体)50μg、または、control IgG(rabbit IgG)50μgを、BALB/cマウスの腹腔内へ投与した。 Twenty-four hours before the sixth, ninth and twelfth administration of OVA, 50 μg of an anti-DOCK8 antibody derived from rabbit (the same antibody used for [Test 1]) or 50 μg of control IgG (rabbit IgG) was added. It was administered intraperitoneally to BALB / c mice.
12回目のOVAの投与後に採血を行い、血清中の自己抗体(具体的には、RF(rheumatoid factor)、anti-Sm Ab、および、anti-dsDNA Ab)をELISA(enzyme-linked immunosorbent assay)により検出した。 Blood was collected after the 12th OVA administration, and autoantibodies (specifically, RF (rheumatoid factor), anti-Sm Ab, and anti-dsDNA Ab) in serum were measured by ELISA (enzyme-linked immunosorbent assay). Detected.
下記の表2にELISAの試験結果を示す。 Table 2 below shows the ELISA test results.
また、表2に示すように、6回目、9回目および12回目のOVAの投与前にcontrol IgGを投与したBALB/cマウス群(表2の「OVA×12 + control IgG」)と比較して、6回目、9回目および12回目のOVAの投与前に抗DOCK8抗体を投与したBALB/cマウス群(図3の「OVA×12 + anti-DOCK8 Ab」)では、3種類の自己抗体の全てが、有意に減少した。 In addition, as shown in Table 2, compared with the BALB / c mouse group (“OVA × 12 + control IgG” in Table 2) to which control IgG was administered before the 6th, 9th, and 12th OVA administrations. , All three types of autoantibodies in the BALB / c mouse group (“OVA × 12 + anti-DOCK8 Ab” in FIG. 3) to which the anti-DOCK8 antibody was administered before the 6th, 9th and 12th OVA administrations. However, it decreased significantly.
BALB/cマウス(Charles River Japan Inc.)に、卵白アルブミン(OVA:grade V; Sigma)0.5mgを、5日毎に繰り返し腹腔内投与した。これによって、自己免疫疾患の症状を呈するマウスを作製した。
BALB / c mice (Charles River Japan Inc.) were repeatedly intraperitoneally administered 0.5 mg of ovalbumin (OVA: grade V; Sigma) every 5 days. As a result, mice exhibiting symptoms of autoimmune disease were produced.
12回目のOVAの投与の後、T細胞を幾つかの亜集団に分画した。周知の方法にしたがって、各亜集団のT細胞から、細胞質画分と、膜画分とを取得した。 After the 12th OVA administration, T cells were fractionated into several subpopulations. Cytoplasmic fractions and membrane fractions were obtained from T cells of each subpopulation according to a well-known method.
1レーンあたり10μgの細胞質画分、および、1レーンあたり10μgの膜画分を電気泳動(10%ポリアクリルアミドゲル)に供し、各画分に含有されるタンパク質を分離した。抗DOCK8抗体を用いたウエスタンブロット法にて、分離されたタンパク質に含まれているDOCK8タンパク質を検出した。その結果を図2に示す。 A cytoplasmic fraction of 10 μg per lane and a membrane fraction of 10 μg per lane were subjected to electrophoresis (10% polyacrylamide gel), and the proteins contained in each fraction were separated. The DOCK8 protein contained in the separated protein was detected by Western blotting using an anti-DOCK8 antibody. The results are shown in FIG.
図2において、レーン1および5は、各々、Whole T細胞に由来する、細胞質画分および膜画分の試験結果である。また、レーン2および6は、各々、CD45RBhigh122high細胞に由来する、細胞質画分および膜画分の試験結果である。また、レーン3および7は、各々、PD-1-CD45RBlow122low細胞に由来する、細胞質画分および膜画分の試験結果である。また、レーン4および8は、各々、PD-1+CD45RBlow122low細胞に由来する、細胞質画分および膜画分の試験結果である。In FIG. 2,
これらの細胞のうち、PD-1+CD45RBlow122low細胞に、自己応答性・自己抗体誘導性CD4 T細胞(autoantibody-inducing CD4 T cells;aiCD4 T細胞)が含まれている。また、図2において、略230kDaの位置に観察される像が、DOCK8タンパク質に対応する。Among these cells, PD-1 + CD45RB low 122 low cells contain autoantibody-inducing CD4 T cells (aiCD4 T cells). Also, in FIG. 2, the image observed at a position of approximately 230 kDa corresponds to the DOCK8 protein.
図2のレーン4および8から明らかなように、PD-1+CD45RBlow122low細胞では、細胞質画分のみならず、膜画分に多くのDOCK8タンパク質が含まれていた。このことは、D-1+CD45RBlow122low細胞では、細胞膜の表面に多くのDOCK8タンパク質が局在していることを示している。なお、抵DOCK8抗体を用いたフローサイトメトリーによってPD-1+CD45RBlow122low細胞を認識できることからも、D-1+CD45RBlow122low細胞では、細胞膜の表面に多くのDOCK8タンパク質が局在していると考えられる。As is clear from
これまでの研究から、CD4 T細胞亜集団(具体的には、PD-1+CD45RBlow122low)に含まれるaiCD4 T細胞が、多様な自己抗体を誘導するとともに、細胞傷害性T細胞(CTL)を成熟させて種々の臓器障害を惹起して、SLEを発症させることが知られている。それ故に、本試験結果は、細胞質画分に存在するDOCK8タンパク質のみならず、細胞膜の表面に存在するDOCK8タンパク質が、SLEの発症メカニズムに関与していることを示唆している。From previous studies, aiCD4 T cells contained in the CD4 T cell subpopulation (specifically, PD-1 + CD45RB low 122 low ) induce various autoantibodies and cytotoxic T cells (CTL). ) Is matured to cause various organ disorders and cause SLE. Therefore, the results of this test suggest that not only the DOCK8 protein present in the cytoplasmic fraction but also the DOCK8 protein present on the surface of the cell membrane is involved in the pathogenic mechanism of SLE.
本発明の一態様は、自己免疫疾患の予防および/または治療に用いることができる。 One aspect of the invention can be used for the prevention and / or treatment of autoimmune diseases.
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